Genetic transformation of crop plants with heterologous genes is widely recognized as an effective strategy for the introduction of desirable traits such as male sterility/restoration for hybrid seed production, insect resistance, nutritional enhancement, resistance to biotic and abiotic stresses, etc. in crop plants. Expression levels of the transgene(s) are critical for achieving the desired phenotype in transgenic plants. Transgene expression levels can be substantially enhanced by modifications at the transcriptional level (by the use of strong promoters/enhancers) or at the translational level (by modulating parameters viz., codon usage, mRNA stability, etc. associated with coding regions of transgenes). However, the existence of various regulatory mechanisms in plants renders the introduced transgene(s) susceptible to down-regulation which, in many instances, also leads to transgene silencing. Homology based gene silencing (HBGS) has been reported to occur extensively in transgenic plants (Meyer and Saedler 1996, Ann. Rev. Plant Physiol. Plant Mol. Biol. 47:23-48; Vaucheret and Fagard 2001, Trends Genet. 17:29-35) and can occur at the transcriptional (transcriptional gene silencing, TGS) as well as at the post transcriptional levels (post transcriptional gene silencing, PTGS). Such situations can arise when (i) multiple copies of a gene cassette integrate into plants during transformation (van der Krol et al. 1990, Plant Cell 2:291-299), (ii) introduced transgenes are transcribed using homologous promoters (Mol et al. 1989, Plant Mol. Biol. 13:287-294) or (iii) an introduced gene has homology with an endogenous gene in the coding region (Napoli et al. 1990, Plant Cell 2:279-289). Several mechanisms have been suggested to explain the phenomena of HBGS (Matzke and Matzke 1995, Plant Physiol. 107:679-685; Meyer and Saedler 1996, Ann Rev. Plant Physiol Plant Mol. Biol. 47:23-48; Fire 1999, Trends Genet. 15:358-363; Hamilton and Baulcombe 1999, Science 286:950-952; Steimer et al. 2000, Plant Cell 12:1165-1178), the common denominator being that the homology triggers cellular recognition mechanisms that result in silencing of the repeated genes. The present invention relates to a method for enhancing expression levels of transgene(s) while reducing its susceptibility to homology based post transcriptional gene silencing. The strategy of the present invention has been tested by enhancing expression of a restorer gene (barstar) to facilitate effective restoration of male fertility in barnase-containing male sterile transgenic plants for hybrid seed production in crop plants.
The contributions of hybrids towards enhancing crop productivity are well recognized. Increase in crop productivity is due to the phenomenon of heterosis or hybrid vigor in which F1 hybrid plants generated by crosses between two genetically diverse parents exhibit an improved yield than either of the two parents (Banga 1992, In Breeding Oilseed Brassicas Eds. Labana K S, Banga S S and Banga S K, Narosa Publishing House, New Delhi; Pradhan et al 1993, Euphytica 69:219-229). Cross pollination is essential for the production of hybrids. In normally self-pollinating crop plants (for example, Brassica sp., rice and wheat), one of the parents needs to be male sterile to facilitate cross pollination. In crop plants wherein seeds are the desired economic products, availability of a suitable restorer system in the male parent is essential in order to achieve seed set in the F1 hybrids.
One of the approaches for generation of male sterile/restorer lines for hybrid seed production is the use of cytoplasmic male sterility (CMS)/restorer systems. CMS is a maternally inherited phenomenon, the genetic determinants of which are located in genomes of the cytoplasmic organelles, the mitochondria. Such plants are severely impaired in their ability to produce functional pollen grains. Restorer genes for CMS systems are dominant nuclear genes that suppress male sterile effects of the cytoplasm (i.e., mitochondria). On being incorporated into the male parent, they can be used as restorers of male fertility in F1 hybrids. CMS systems have been widely used in the production of hybrids in sorghum, sunflower, pearl millet and sugar beet. However, their use has been limited in corn, wheat and oilseed Brassicas due to linkage of undesirable traits such as increased disease susceptibility, chlorosis, distortion of petals, poor nectary function, etc. with CMS in these systems (McVetty et al 1989, Can J. Plant Sci. 69:915-918; Burns et al 1991, Can. J. Plant Sci. 71:655-661; Williams 1995, Trends Biotech. 13:344-349).
With the advent of recombinant DNA and plant transformation technologies, genetic engineering of male sterility and its restoration have emerged as tangible options for the development of male sterile and restorer lines for hybrid seed production. Male sterility can be artificially induced in plants by introducing a male sterility gene comprising a DNA sequence coding, for example, a cytotoxic product. The cytotoxic product may be a lethal gene under transcriptional control of a promoter which is predominantly active in selective tissue(s) of male reproductive organs in plants. Tapetum, which forms the innermost layers of the anther wall, is one of the most important tissues associated with pollen development. Disruption of tapetal cells by the expression of toxic proteins would therefore impair pollen development leading to male sterile plants. Male sterility was successfully induced in transgenic tobacco and oilseed rape (Brassica napus) by targeted expression of a ribonuclease [barnase from Bacillus amyloliquefaciens (Hartley 1989, Trends Biochem. Sciences 14:450-454) or Rnase T1 from Aspergillus oryzae] in the tapetal tissues using a tapetum-specific promoter (TA29) from tobacco (Mariani et al 1990, Nature 347:737-741).
Presence of a selectable marker gene in male sterility inducing constructs is also essential to facilitate in vitro selection of transformants and for field selection of plants containing the male sterility gene. For example, the bar gene from Streptomyces hygroscopicus, conferring resistance to the herbicide, Basta can be used as an in vitro as well as field selectable marker. Use of a strong constitutive promoter to express the marker gene is important for efficient selection of transformants. However, in a study on development of male sterile barnase lines in Brassica juncea (Indian mustard) (Jagannath et al 2001, Mol. Breeding 8:11-23), it was found that tissue-specific expression of the barnase gene was deregulated in the presence of a strong constitutive promoter (CaMV35S) that was used to express the herbicide resistance-conferring selectable marker gene (bar) in barnase constructs. Deregulated expression of the cytotoxic barnase gene not only reduced the recovery of transgenic shoots in transformation experiments, but also affected agronomically important traits viz., vegetative morphology, female fertility, seed germination frequencies and inheritance of male sterility in barnase lines rendering them unsuitable for agronomic applications. To circumvent this problem, the above study described a method of using a Spacer DNA fragment to protect tissue-specific expression of the barnase gene which substantially enhanced the recovery of agronomically viable male sterile lines in Brassica juncea. Further, all male sterile lines developed in this study were found to be stable over several generations under field conditions in contrast to those developed earlier (Mariani et al 1990, Nature 347:737-741) in which several barnase lines showed breakdown of the male sterile phenotype even under controlled growth conditions (Denis et al 1993, Plant Physiol. 101:1295-1304).
Barnase activity in B. amyloliquefaciens is inhibited by a specific inhibitor, barstar, which negates the lethal effects of the ribonuclease by forming a one-to-one complex with the same in the cytoplasm (Hartley and Smeaton 1973, J. Biochem. 248:5624-5626). Expression of the barstar gene under the TA29 tapetum-specific promoter (which is also used for expression of the baniase gene to develop male sterile plants) was shown to restore fertility in barnase lines of Brassica napus (Mariani et al 1992, Nature 357:384-387). In the above study, four transgenic barstar lines containing a single copy of the T-DNA insert were crossed to four single-copy male sterile barnase lines in different combinations and resulting F1 hybrids were analyzed for restoration of male fertility in the same. It was found that six of nine crosses performed between barnase and barstar lines produced male fertile progeny while two crosses failed to restore fertility. In one case, partial restoration of fertility was also seen. Inability of some barstar lines to restore fertility was attributed to insufficient levels of the inhibitor protein due to its poor expression. However, the above study was found to be lacking in the analysis of pollen viability in restored plants. Hence, it is not possible to ascertain the extent of fertility restoration in male fertile progeny. This is of particular significance in case of oilseed crops such as Brassicas wherein seed set is of paramount importance in achieving full yield potential of the crop.
Plant Genetic Systems have described, in EP 0412911 A1, use of the barstar gene for restoration of fertility in male sterile barnase lines. The above patent describes development of restorer lines in tobacco and oilseed rape using a DNA construct containing three chimeric sequences (i) the barstar gene under transcriptional control of the TA29 tapetum-specific promoter (ii) a selectable marker gene (neo, conferring kanamycin resistance) expressed using a suitable constitutive promoter (pNos) for in vitro selection of transformants and (iii) another selectable marker gene (sfr) expressed using the constitutive Rubisco ssu promoter from Arabidopsis. The above patent also describes various breeding strategies which can be used for hybrid seed production using the barnase/barstar system of male sterility and restoration. However, no experimental evidence has been provided for any of these strategies and therefore, the efficacy of the same cannot be conclusively established.
Another strategy for the generation of male sterile and restorer lines using the barnase/barstar system is described in U.S. Pat. No. 5,929,307 wherein the FLP/FRT recombinase system of yeast is used to regulate expression of the barnase and barstar genes. According to one of the embodiments of this invention, a male fertile transgenic plant can be generated by transformation using a vector containing a cytotoxic gene (barnase) and a restorer gene (barstar) wherein the restorer gene is flanked by site-specific (first) recombination sites which are recognized by a specific (first) recombinase. The male sterility and restorer genes are under independent transcriptional control of an anther-specific promoter. Further, the entire functional element composed of the above two genes and their respective promoters is flanked by another set of (second) site-specific recombination sites which are recognized by a specific (second) recombinase. A male sterile plant can be produced by crossing the above male fertile plant with a transgenic plant containing the first site-specific recombinase expressed under a constitutive promoter. Alternatively, the DNA construct used to generate the male fertile plant may also contain the first recombinase expressed under an inducible promoter, in which case, male sterility can be achieved by inducing expression of the first recombinase. Restoration of fertility can be achieved in either case by crossing the male sterile plant with a transgenic plant containing a gene encoding the second recombinase. The invention also describes several other alternative embodiments based on the use of site-specific recombinase systems to generate male sterile lines and for restoration of fertility in the same. However, data presented in the above study only establishes functionality of the yeast FLP/FRT system in maize cells and transformed Arabidopsis thaliana plants using various reporter genes. No experimental evidence is presented in support of functionality of the system in induction of male sterility and its restoration based on various strategies described in the same.
Novartis Finance Corporation has described, in U.S. Pat. No. 6,147,282, another strategy for controlling plant fertility based on chemical control of gene expression in plants. According to this strategy, chemical ligands can be used to activate receptor polypeptides (for example, the Ecdysone and USP receptors containing ligand-binding and DNA-binding domains) which, in turn, induce expression of a target gene (barnase or boustar) expressed using a promoter containing suitable receptor binding elements. According to the patent, the above strategy could be used for induction of male sterility as well as its restoration. Male sterile transgenic plants can be generated using a DNA construct containing: (i) the barnase gene expressed under suitable anther-specific promoters (for example, TA29) (ii) the receptor expression cassettes (encoding the Ecdysone and USP receptor polypeptides) under regulatory control of the same anther specific promoter or from constitutive promoters (for example, maize ubiquitin, 35S or rice actin) and (iii) a target expression cassette having a 5′ regulatory element (containing the appropriate response element sequence and core promoter elements) linked to the barstar gene. In order to restore fertility, a suitable chemical ligand (for example, RH5992) can be sprayed on the male sterile transgenic plants. This would induce heterodimerization of the USP and Ecdysone receptor polypeptides resulting in the activation of the 5′ regulatory sequence of the target (barstar) expression cassette and production of barstar protein which would inhibit barnase and restore male fertility. The functionality of the above strategy was demonstrated using the luciferase reporter gene as the target polypeptide. Expression of luciferase protein was reported to increase approximately 2-4 folds upon induction over that observed in the absence of the chemical ligand. However, in the absence of corresponding data on expression of barstar protein for restoration of male fertility and in light of published literature on problems associated with development of male sterile lines due to deregulated expression of cytotoxic gene(s) (see above), efficiency of the above strategy in achieving the desired fertility status in crop plants needs to be established. Moreover, this strategy requires exogenous application of chemicals to induce the desired fertility status. For crop plants grown on a large acreage, this might not only be economically unsound, but the duration, time and amount of the desired ligand to be sprayed on standing crops would also be highly variable leading to inconsistency in production parameters.
Transgene expression levels can be enhanced by modification of the coding sequence to introduce preferentially used codons (for improved mRNA translation) and to ensure removal of potential deleterious signals which might inhibit transgene expression. Modified genes have been shown to improve transgene expression levels in heterologous systems including bacteria, plants and animal cell lines (Wosnick et al 1989, Gene 76:153-160; Adams et al 1988, Nucl. Acids Res. 16:4287-4298; Perlak et al 1991, PNAS-USA 88:3324-3328; Koziel et al 1993, BioTechnology 11:194-200; Pang et al 1996, Plant Physiol. 105:473-482). Common factors which have been taken into consideration for designing modified genes for improved expression in plant systems include codon usage, mRNA instability sequences (for example, polyadenylation signals), GC-content and plant intron consensus sequences. The cry genes of Bacillus thuringiensis, encoding the insecticidal-δ-endotoxins, are some of the best representative examples of bacterial genes modified for enhanced expression in transgenic plants (Perlak et al 1991, PNAS-USA 88:3324-3328; Adang et al 1993, Plant Mol. Biol. 21:1131-1145; Fujimoto et al 1993, BioTechnology 11:1151-1155; Strizhov et al 1996, PNAS-USA 93:15012-15017; Iannaocone et al 1997, Plant Mol. Biol. 34:485-496). However, in a study by Rouwendal et al (1997) on the wild type and modified versions of the Green Fluorescent Protein (GFP) gene, no enhancement in expression levels could be achieved using modified transgenes in comparison to that obtained using the wild type sequence (Rouwendal et al 1997, Plant Mol. Biol. 33:989-999). In another study on the luciferase gene, it was reported that while the modified gene sequence may enhance expression of the transgene in one plant system, it may not do so in others (Lonsdale et al 1998, Plant Cell Rep. 17:396-399).
Plant Genetic Systems, in WO9810081, has described modification of the barstar gene sequence for enhanced expression in plant cells. According to the invention, the modified barstar DNA could be used for the development of efficient restorer lines which are capable of restoring fertility to a variety of male sterile lines (with varying levels of barnase expression) and are particularly useful for producing restorer lines in cereals, especially in corn, rice and wheat. The improved barstar gene sequence described in this invention encodes a barstar protein with an altered N-terminal amino acid sequence that starts with Methionine followed by “X” amino acid wherein “X” is either Alanine, Valine, Glycine, Aspartic acid or Glutamic acid, preferably Alanine encoded by a GCC codon. The preference for these amino acids is because, all available codons of these amino acids begin with a G nucleotide and being the second amino acid in the modified barstar gene sequence, it would provide an optimal translation initiation context at position +4. The modified barstar gene of the invention has an amino acid sequence which is “Met-Ala-Lys” while the wild type protein begins with “Met-Lys” at the N-terminal end. Other criteria used for designing the modified barstar gene sequence (also referred to as “synthetic barstar DNA”) of the above invention and incorporated therein were:    (a) Codon usage: The modified sequence has a codon usage that is optimized for oilseed rape, cotton, maize, rice and wheat, preferably for oilseed rape (Brassica napus), maize and rice. For the purpose of this invention, the overall codon frequencies for various plant species described by Ikemura were used (Ikemura 1993, In “Plant Molecular Biology Labfax”, Croy ed., Bios Scientific Publishers Ltd., pp. 37-48). According to this invention, the preferred codon selected for incorporation in the modified barstar sequence is one, which, in each of more than N/2 plant species (N=No. of plant species for which codon usage patterns are taken into account for design of the modified sequence) has an overall frequency that is at least twice the overall frequency of the least used codon and/or more than half of the overall frequency of the most used codon    (b) AT-content: A and T nucleotides constitute less than 40% of the sequence composition. This was primarily done to reduce the probability of introducing polyadenylation signals and intron recognition sequences which are known to destabilize transgene expression and can occur with a greater probability in AT-rich sequences. The synthetic barstar sequence of the invention has an AT-content of 38.4% while the wild type sequence is characterized by an AT-content of 51.6%.    (c) CG and CNG sequences: CG and CNG sequences have been shown to be associated with DNA methylation and gene silencing in plants. The modified sequence should not contain more than 7% CG dinucleotides and/or no more than 9.5% of CNG trinucleotides. The synthetic barstar DNA of the above invention contains 16 CG dinucleotides and 24 CNG trinucleotides while the wild type sequence has 14 CGs and 22 CNGs.    (d) The modified sequence is further characterized by having no more than 7, preferably no more than 5, particularly no more than 3 tetranucleotides consisting of only one kind of nucleotide and having no more than 2, preferably no more than 1 pentanucleotide consisting of only one kind of nucleotide. The synthetic barstar DNA of the invention contains one CCCCC and one GGGG stretch.
The improved barstar gene was deployed for the development of restorer lines in rice, corn and oilseed rape. In transgenic rice plants generated using the wild type and synthetic barstar genes under regulatory control of appropriate male tissue-specific promoters, it was found that the synthetic gene produced more barstar protein with the amount of barstar protein being proportional to the amount of barstar mRNA present. Crosses between male sterile barnase lines and restorer barstar plants showed that restoration capabilities were directly comparable with the amount of barstar protein produced. Plants producing 50 ng or more barstar/mg of extracted protein functioned as efficient restorer lines while plants producing between 4-10 ng barstar/mg of extracted protein could only induce partial restoration of male fertility. While plants with barstar expression levels >50 ng/mg of total protein could be obtained with the synthetic barstar gene, none of the plants generated using the wild type gene gave comparable expression levels. Similar studies were performed on transgenic corn plants. One male sterile line (designated MS3), which, according to the inventors, was a considerably difficult line to restore (data not shown), was selected for testing restoration capabilities of restorer lines generated using the wild type and synthetic barstar genes. Crosses between the male sterile MS3 line and seven wild type barstar restorer lines did not identify any suitable restorer, with maximally 75% of anthers producing viable pollen. On testing the MS3 line with restorer lines generated using the synthetic barstar sequence, it was found that four out of six lines tested were able to achieve complete restoration of male fertility thereby demonstrating that restorer lines generated using the synthetic barstar gene have a significantly better restoration capacity. The inventors further report that the amount of barstar protein in two restorer plants developed using the synthetic barstar DNA was much higher (210 and 100 ng barstar/mg total protein) than that observed in one wild type barstar plant (20 ng barstar/mg total protein). Transgenic plants were also generated in oilseed rape using the wild type and synthetic barstar genes. Analysis of activity of the wild type and synthetic barstar proteins revealed that activity of the improved barstar protein was at least equivalent to that of its wild type counterpart. However, no data were reported on restoration capabilities of these barstar lines. It is also important to note here that the above studies are lacking in data on copy numbers of the integrated transgene for the experimental lines used. Therefore, it is probable that position effects and varying copy numbers of the barstar gene may be influencing barstar expression levels in independent transgenic plants. Moreover, in the absence of data on pollen viability in restored plants, extent of restoration cannot be determined accurately.
Several strategies have been developed for generation of restorer lines for hybrid seed production. One of these studies has also addressed the issue of expression level of the restorer gene product and has prescribed the design and use of synthetic DNA sequences for enhanced expression in heterologous systems to facilitate effective restoration. However, none of the described strategies highlight the extent of restoration with reference to pollen viability which is a critical parameter for determining the efficacy of any restoration strategy. Additionally, most strategies rely on the use of several superfluous genes, promoters and other DNA sequences, the presence of which is highly undesirable in transgenic crop systems.
The present invention describes a method for enhancing expression levels of transgene(s) while reducing its susceptibility to homology based post transcriptional gene silencing. It further describes a method for achieving stable, enhanced and extended temporal expression of a restorer gene product based on the simultaneous use of two different restorer gene sequences encoding the same protein product in the same DNA construct. One of the said sequences is modified for expression in dicotyledonous crop plants using codon degeneracy to avoid homology between the two sequences at the DNA and mRNA levels and the other is the naturally occurring wild type sequence, each of which is placed under independent transcriptional control of regulatory elements with overlapping expression patterns in male reproductive tissues. In addition to enhancing restorer gene expression, simultaneous use of two different DNA sequences (which would transcribe two different RNA molecules) in the same DNA construct confers the added advantage of avoiding homology based post transcriptional gene silencing. The method described in the present invention can also be applied with suitable modifications to other molecular methods based on the expression of a cytotoxic gene and its corresponding inhibitor for the development of male sterile and restorer lines, respectively.